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Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

Objective To investigate the durability of the first integrase inhibitor-based regimen in a HIV geriatric multicentric prospective cohort and to explore the reasons of regimen discontinuation. Design This is an analysis conducted on the Geriatric Patients Living with HIV/AIDS (GEPPO) cohort, an Italian prospective observational multicentre cohort of people living with HIV with 65 years of age or more. Methods The analysis was performed using R (version 4.0.2). The tests performed were two sided assuming a 5% significance level (Kruskal-Wallis test, Chi-squared test, log-rank test and a Cox Proportional Hazard model). The proportion of participants discontinuing the three regimens was displayed using cumulative curves. Results Among 1531 patients enrolled between 2017 and 2019 in the GEPPO cohort, we included 822 participants in this analysis. At baseline, median age was 69.8, the immunovirological profile good, multimorbidity was present in 42.3% of participants, while 27.4% were on polypharmacy. Overall, 483, 243 and 96 participants received DTG, RAL and EVG/c respectively as first InSTI. At the end of the follow up 6.4%, 21.1% and 22.9% participants discontinued DTG, RAL and EVG/c respectively. Using a log-rank test, EVG showed a significantly lower durability than DTG (p Conclusions The three integrase inhibitors considered showed a good durability and no virological failures in geriatric patients such as those enrolled in the GEPPO cohort when used in a two or three drug regimen.

Prostaglandin E2(PGE2)-induced coughsin vivoand vagal nerve depolarizationin vitroare inhibited by systemic and local administration of prostaglandin EP3 receptor (L-798106) and TRPV1 antagonists (JNJ 17203212). These results indicate a modulating effect of TRPV1 on the EP3 receptor-mediated cough responses to PGE2likely through the vagal sensory nerve. This study aimed to determine whether 1) inhalation of aerosolized JNJ 17203212 and L-798106 affected cough responses to citric acid (CA, mainly stimulating TRPV1) and PGE2; 2) TRPV1 and EP3 receptor morphologically are co-expressed and electrophysiologically functioned in the individual of vagal pulmonary C-neurons (cell bodies of bronchopulmonary C-fibers in the nodose/jugular ganglia); and 3) there was a cross-effect of TRPV1 and EP3 receptor on these neural excitations. To this end, aerosolized CA or PGE2was inhaled by unanesthetized guinea pigs pretreated without or with each antagonist given in aerosol form. Immunofluorescence was applied to identify the co-expression of TRPV1 and EP3 receptor in vagal pulmonary C-neurons (retrogradely traced by DiI). Whole-cell voltage patch clamp approach was used to detect capsaicin (CAP)- and PGE2-induced currents in individual vagal pulmonary C-neurons and determine the effects of the TRPV1 and EP3 receptor antagonists on the evoked currents. We found that PGE2-induced cough was attenuated by JNJ 17203212 or L-798106 and CA-evoked cough greatly suppressed only by JNJ 17203212. Approximately 1/4 of vagal pulmonary C-neurons co-expressed EP3 with a cell size < 20 μm. Both CAP- and PGE2-induced currents could be recorded in the individuals of some vagal pulmonary C-neurons. The former was largely inhibited only by JNJ 17203212, while the latter was suppressed by JNJ 17203212 or L-798106. The similarity of the cross-effect of both antagonists on cough and vagal pulmonary C-neural activity suggests that a subgroup of vagal pulmonary C-neurons co-expressing TRPV1 and EP3 receptor is, at least in part, responsible for the cough response to PGE2.

Therapeutic drug monitoring (TDM) is used in certain clinically selected cases and in research settings to optimize the response to antiretroviral therapy. Plasma of blood is commonly used for TDM, but blood sampling is invasive and at risk for transmission of infectious agents. On the other hand, saliva sampling is noninvasive, safe, cheap, and easily performed compared to blood. Dolutegravir (DTG) is now widely prescribed as a key component of antiretroviral therapy for HIV infection. In this study, we examined the relationship between DTG concentrations in plasma and saliva of treated patients to explore the possibility of using saliva as an alternative body fluid of TDM. A total of 17 pairs of blood and saliva samples were obtained from 15 consented HIV-1-infected subjects treated with DTG containing regimens for more than one month. Both blood and saliva samples were collected within 1 h of each other. Drug concentrations were determined by liquid chromatography-tandem mass spectrometry using DTG-d5 as an internal standard. The LLOQ was 0.5 ng/mL. The calibration curves were prepared with pooled plasma or saliva containing DTG in a range of 0.5–100 ng/mL with precision of r = 0.76, p < 0.001). The median ratio of the drug concentration in saliva to those in plasma was 0.0056, which is close to the rate of non-protein-bound DTG in plasma (0.70%), suggesting that only free DTG in plasma is transported to the salivary glands and secreted into saliva. The present study demonstrates that DTG concentration in saliva reflects the pharmacologically active drug concentration in plasma and may provide an easily accessible alternative for monitoring effective antiretroviral treatment.

Theoretically, small molecule CDK4/6 inhibitors (CDK4/6is) represent a logical therapeutic option in non-small cell lung cancers since most of these malignancies have wildtype RB, the key target of CDKs and master regulator of the cell cycle. Unfortunately, CDK4/6is are found to have limited clinical activity as single agents in non-small cell lung cancer. To address this problem and to identify effective CDK4/6i combinations, we screened a library of targeted agents for efficacy in four non-small cell lung cancer lines treated with CDK4/6 inhibitors Palbociclib or Abemaciclib. The pan-PAK (p21-activated kinase) inhibitor PF03758309 emerged as a promising candidate with viability ratios indicating synergy in all 4 cell lines and for both CDK4/6is. It is noteworthy that the PAKs are downstream effectors of small GTPases Rac1 and Cdc42 and are overexpressed in a wide variety of cancers. Individually the compounds primarily induced cell cycle arrest; however, the synergistic combination induced apoptosis, accounting for the synergy. Surprisingly, while the pan-PAK inhibitor PF03758309 synergizes with CDK4/6is, no synergy occurs with group I PAK inhibitors FRAX486 or FRAX597. Cell lines treated only with Ribociclib, FRAX486 or FRAX597 underwent G1/G0 arrest, whereas combination treatment with these compounds predominantly resulted in autophagy. Combining high concentrations of FRAX486, which weakly inhibits PAK4, and Ribociclib, mimics the autophagy and apoptotic effect of PF03758309 combined with Ribociclib. FRAX597, a PAKi that does not inhibit PAK4 did not reduce autophagy in combination with Ribociclib. Our results suggest that a unique combination of PAKs plays a crucial role in the synergy of PAK inhibitors with CDK4/6i. Targeting this unique PAK combination, could greatly improve the efficacy of CDK4/6i and broaden the spectrum of cancer treatment.

Sirodesmin, the major secondary metabolite produced by the plant pathogenic fungus Leptosphaeria maculans in vitro, has been linked to disease on Brassica species since the 1970s, and yet its role has remained ambiguous. Re-examination of gene expression data revealed that all previously described genes and two newly identified genes within the sir gene cluster in the genome are down-regulated during the crucial early establishment stages of blackleg disease on Brassica napus. To test if this is a strategy employed by the fungus to avoid damage to and then detection by the host plant during the L. maculans asymptomatic biotrophic phase, sirodesmin was produced constitutively by overexpressing the sirZ gene encoding the transcription factor that coordinates the regulation of the other genes in the sir cluster. The sirZ over-expression strains had a major reduction in pathogenicity. Mutation of the over-expression construct restored pathogenicity. However, mutation of two genes, sirP and sirG, required for specific steps in the sirodesmin biosynthesis pathway, in the sirZ over-expression background resulted in strains that were unable to synthesize sirodesmin, yet were still non-pathogenic. Elucidating the basis for this pathogenicity defect or finding ways to overexpress sirZ during disease may provide new strategies for the control of blackleg disease.

Triple negative breast cancer (TNBC) represents approximately 10–15% of all breast cancers and has a poor outcome as it lacks a receptor target for therapy, and TNBC is frequently associated with a germline mutation of BRCA1. Poly (ADP-ribose) polymerase inhibitor (PARPi) drugs have demonstrated some effectiveness in treating BRCA1 or BRCA2 mutated breast and ovarian cancers but resistance to PARPi is common. Published results found that resistance to Olaparib, a PARPi, can be due to downregulation of EMI1 and the consequent upregulation of the RAD51 recombinase. Using a tissue culture-based cell viability assay, we extended those observations to another PARPi and to other chemotherapy drugs that affect DNA repair or the cell cycle. As we expected, EMI1 downregulation resulted in resistance to another PARPi drug, Talazoparib. EMI1 downregulation also led to resistance to other cytotoxic drugs, Cisplatin and CHK1 inhibitor. Surprisingly, EMI1 depletion also led to resistance to a MEK inhibitor, though this inhibitor blocks cells in G1 phase of the cell cycle and would not be expected to be sensitive to EMI1 levels. Notably, increasing the RAD51 protein expression only partially recapitulated the effects of EMI1 depletion in causing resistance to different PARPi and the other cytotoxic drugs. These results suggest that the downstream effects of EMI1 downregulation that contribute to PARPi resistance are increasing the concentration of RAD51 protein in the cell and blocking mitotic entry. We found that combining CHK1 inhibitor with olaparib results in restoration of sensitivity even when EMI1 expression is downregulated. This combination therapy may be a means to overcome the PARPi resistance in BRCA1-deficient TNBC cells.

BACKGROUND: Multiple T-cell marker recovery (MTMR: CD4+ T-cells >500 cel/mm3 plus CD4+% >29% plus CD4+/CD8+ ratio >1) has been proposed as the most complete level of immune reconstitution. In this study we quantified differences in the CD4+/CD8+ ratio, CD4+% recovery and MTMR after starting HIV-1 treatment with dolutegravir (DTG) vs. raltegravir (RAL) plus a NRTI backbone. METHODS: Exploratory post-hoc analysis of the SPRING-2 study, a randomized double-blind clinical trial comparing DTG and RAL as third agents in naive HIV-infected patients at 100 sites in Canada, USA, Australia, and Europe. Percentage differences and corresponding precision based on 95% confidence intervals (CI) and p-values were calculated for i) CD4+/CD8+ ratio normalization, ii) CD4+% normalization, and iii) the achievement of MTMR. RESULTS: A total of 822 participants were analyzed (411 in each group). No statistically significant differences in the proportion of patients who reached a CD4+/CD8+ ratio ≥0.5 & ≥1 at w48 & w96 were observed. At w96, the proportion of patients with a CD4+/CD8+ ratio ≥1 was similar (30.43% DTG vs. 29.57% RAL). No differences were observed in the mean increase in CD4+/CD8+ ratio from baseline at both w48 & w96. Similarly, no significant differences in the CD4+/CD8+>29% were observed at w96 (72.95% DTG vs 69.28% RAL). The proportion of patients attaining MTMR criteria was also similar in the DTG group and the RAL group at w48 (20.33% vs. 18.26%; difference 2.07 (95%CI (-3.67;7.81) P = 0.481 and w96 (28.70% vs. 27.13; difference 1.56 (95%CI -5.22;8.34) P = 0.652). CONCLUSION: After comparing DTG and RAL, no differences on immune recovery markers were observed. This work has been (partially) funded by the SPANISH AIDS Research Network (RIS) (RD16/0025/0001, RD16/0025/0036 and RD16CIII/0002/0006) and project as part of the Plan Nacional R+D+I and cofinanced by ISCIII- Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study. The authors received no specific funding for this work. Sí

Resistance associated mutations (RAMs) threaten the long-term success of combination antiretroviral therapy (cART) outcomes for HIV-1 treatment. HIV-1 Integrase (IN) strand transfer inhibitors (INSTIs) have proven to be a viable option for highly specific HIV-1 therapy. The INSTI, Dolutegravir is recommended by the World Health Organization for use as first-line cART. This study aims to understand how RAMs affect the stability of IN, as well as the binding of the drug Dolutegravir to the catalytic pocket of the protein. Molecular modelling of HIV-1C IN was performed using the SWISS-MODEL webserver; with quality assessment performed using internal methods and external software tools. The site directed mutator webserver was used to predict destabilizing and/or stabilizing effects of known RAMs while FoldX confirmed any changes in protein energy upon introduction of mutation. Interaction analysis between neighbouring residues was done using PyMOL. Three randomly selected mutations were chosen for molecular dynamic simulation studies using Gromacs. Trajectory analysis included Root mean square deviation and fluctuation, Radius of gyration, Principal component analysis and Interaction analysis between Dolutegravir and protein residues. The structural quality assessment indicated high reliability of the HIV-1C IN tetrameric structure, with more than 90% confidence in modelled regions. Change in free energy for the G140S mutant indicated a stabilizing effect and simulation analysis showed it to affect structural stability and flexibility of the protein structure. This was further supported by the drug being expelled from the G140S mutant active site, as indicated by interaction analysis. Our findings suggest the G140S mutant has a strong effect on the HIV-1C IN protein structure and Dolutegravir binding and should be validated using laboratory-based experiments. This approach can be applied to determine the effect of other mutations/variants on HIV-1C integrase drug binding.

BackgroundThe ability of heart valve cells to respond to their mechanical environment represents a key mechanism by which the integrity and function of valve cusps is maintained. A number of different mechanotransduction pathways have been implicated in the response of valve cells to mechanical stimulation. In this study, we explore the expression pattern of several mechanosensitive ion channels (MSC) and their potential to mediate mechanosensitive responses of human valve interstitial cells (VIC).MethodsMSC presence and function were probed using the patch clamp technique. Protein abundance of key MSC was evaluated by Western blotting in isolated fibroblastic VIC (VICFB) and in VIC differentiated towards myofibroblastic (VICMB) or osteoblastic (VICOB) phenotypes. Expression was compared in non-calcified and calcified human aortic valves. MSC contributions to stretch-induced collagen gene expression and to VIC migration were assessed by pharmacological inhibition of specific channels.ResultsTwo MSC types were recorded in VICFB: potassium selective and cation non-selective channels. In keeping with functional data, the presence of both TREK-1 and Kir6.1 (potassium selective), as well as TRPM4, TRPV4 and TRPC6 (cationic non-selective) channels was confirmed in VIC at the protein level. Differentiation of VICFB into VICMB or VICOB phenotypes was associated with a lower expression of TREK-1 and Kir6.1, and a higher expression of TRPV4 and TRPC6. Differences in MSC expression were also seen in non-calcified vs calcified aortic valves where TREK-1, TRPM4 and TRPV4 expression were higher in calcified compared to control tissues. Cyclic stretch-induced expression of COL I mRNA in cultured VICFB was blocked by RN-9893, a selective inhibitor of TRPV4 channels while having no effect on the stretch-induced expression of COL III. VICFB migration was blocked with the non-specific MSC blocker streptomycin and by GSK417651A an inhibitor of TRPC6/3.ConclusionAortic VIC express a range of MSC that play a role in functional responses of these cells to mechanical stimulation. MSC expression levels differ in calcified and non-calcified valves in ways that are in part compatible with the change in expression seen between VIC phenotypes. These changes in MSC expression, and associated alterations in the ability of VIC to respond to their mechanical environment, may form novel targets for intervention during aortic valvulopathies.

The integrase inhibitor dolutegravir was included in initial antiretroviral therapy in Brazil in January 2017. Studies have demonstrated that the efficacy and safety of antiretrovirals have improved with the introduction of new classes of antiretrovirals, such as integrase inhibitors. This study aimed to estimate the frequency of individuals with a virologic response by week 24 of antiretroviral treatment and to describe the adverse events of the regimen containing dolutegravir. This was a cohort of people living with HIV followed up at a referral hospital. Patients were included who had initiated their first treatment between January and August 2017. Data were obtained from medical records, the Drug Logistics Management System and from the Laboratory Tests Control System. Two hundred and twenty-two patients were included for the tolerability analysis and one hundred and thirty-seven for the virologic response analysis. The mean age was 34 years, the median time between diagnosis and initiating treatment was 1.9 months and the median time on antiretroviral therapy was 13.2 months. The frequency of adverse events was 10% (95% CI: 7% to 15.2%), of these, amongst the most frequent events, 91% presented gastrointestinal effects, and 47.8% neuropsychiatric. By week 24 the estimated incidence of virologic response was 89.1% (95% CI: 83% to 93.5%), with an increase during the first 6 months in the number of T-CD4 lymphocytes of 50.7 cells/mm 3 (95% CI: 42 to 59.3). Initial antiretroviral regimens containing dolutegravir were well tolerated and effective in viral suppression during the first 24 weeks after initiating treatment. The occurrence of adverse events was low, either mild or moderate.

The bone marrow is a frequent location of primary relapse after conventional cytotoxic drug treatment of human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Because stromal cells have a major role in promoting chemotherapy resistance, they should be included to more realistically model in vitro drug treatment. Here we validated a novel application of the xCELLigence system as a continuous co-culture to assess long-term effects of drug treatment on BCP-ALL cells. We found that bone marrow OP9 stromal cells adhere to the electrodes but are progressively displaced by dividing patient-derived BCP-ALL cells, resulting in reduction of impedance over time. Death of BCP-ALL cells due to drug treatment results in re-adherence of the stromal cells to the electrodes, increasing impedance. Importantly, vincristine inhibited proliferation of sensitive BCP-ALL cells in a dose-dependent manner, correlating with increased impedance. This system was able to discriminate sensitivity of two relapsed Philadelphia chromosome (Ph) positive ALLs to four different targeted kinase inhibitors. Moreover, differences in sensitivity of two CRLF2-drivenBCP-ALL cell lines to ruxolitinib were also seen. These results show that impedance can be used as a novel approach to monitor drug treatment and sensitivity of primary BCP-ALL cells in the presence of protective microenvironmental cells.

Background There is scant data from India on efficacy and safety of palbociclib and ribociclib in routine clinical practice. Methods This retrospective, observational, single institution study included patients with estrogen and/or progesterone receptor positive and human epidermal growth factor receptor 2 (HER2) negative metastatic breast cancers, who received palbociclib or ribociclib with any partner endocrine therapy in any line of treatment between January 2016 and June 2019. Data were analyzed for progression-free survival (PFS), overall survival (OS) and toxicity. Results The study included 101 female patients with median age of 57 (IQR 48–62) years, of whom 80 (79.2%) were postmenopausal, 79 (78.2%) received palbociclib or ribociclib in second- or later-line treatment, 59 (58.4%) received fulvestrant and 41 (40.6%) received an aromatase inhibitor. In first-line treatment, at a median follow-up of 21.7 (0.5–41.9) months, median PFS and OS were 21.1 (95%CI 16.36-not estimable) months and not reached, respectively. In second- or later-line setting, at a median follow-up of 17.2 (0.5–43.7) months, median PFS and OS were 5.98 (95%CI 4.96–7.89) months and 20.2 (95%CI 14.1-not estimable) months, respectively. Grade 3–4 neutropenia and febrile neutropenia were seen in 45 (45.0%) and 9 (9.0%) patients, respectively while dose reduction was required in 32 (31.7%) patients. In multivariable Cox regression analysis, first-line setting (HR 0.49, 95%CI 0.25–0.97, p = 0.043) and ECOG performance status 1 (HR 0.43, 95%CI 0.20–0.91, p = 0.028) were significantly associated with PFS while only ECOG PS 1 was significantly associated (HR 0.04, 95%CI 0.008–0.206, p = 0.000) with OS. Conclusion Palbociclib and ribociclib, when used in routine clinical practice in first or subsequent lines of treatment, resulted in efficacy and toxicity outcomes in concordance with those expected from pivotal trials.

Common scab is a potato disease characterized by the formation of scab-like lesions on the surface of potato tubers. The actinobacterium Streptomyces scabiei is the main causal agent of common scab. During infection, this bacterium synthesizes the phytotoxin thaxtomin A which is essential for the production of disease symptoms. While thaxtomin A can activate an atypical programmed cell death in plant cell suspensions, it is possible to gradually habituate plant cells to thaxtomin A to provide resistance to lethal phytotoxin concentrations. Potato ‘Russet Burbank’ calli were habituated to thaxtomin A to regenerate the somaclone RB9 that produced tubers more resistant to common scab than those obtained from the original cultivar. Compared to the Russet Burbank cultivar, somaclone RB9 generated up to 22% more marketable tubers with an infected tuber area below the 5% threshold. Enhanced resistance was maintained over at least two years of cultivation in the field. However, average size of tubers was significantly reduced in somaclone RB9 compared to the parent cultivar. Small RB9 tubers had a thicker phellem than Russet Burbank tubers, which may contribute to improving resistance to common scab. These results show that thaxtomin A-habituation in potato is efficient to produce somaclones with increased and durable resistance to common scab.

Within triple negative breast cancer, several molecular subtypes have been identified, underlying the heterogeneity of such an aggressive disease. The basal-like subtype is characterized by mutations in the TP53 gene, and is associated with a low pathologic complete response rate following neoadjuvant chemotherapy. In a genome-scale short hairpin RNA (shRNA) screen of breast cancer cells, polo-like kinase 1 (Plk1) was a frequent and strong hit in the basal breast cancer cell lines indicating its importance for growth and survival of these breast cancer cells. Plk1 regulates progression of cells through the G2-M phase of the cell cycle. We assessed the activity of two ATP-competitive Plk1 inhibitors, GSK461364 and onvansertib, alone and with a taxane in a set of triple negative breast cancer cell lines and in vivo. GSK461364 showed synergism with docetaxel in SUM149 (Combination Index 0.70) and SUM159 (CI, 0.62). GSK461364 in combination with docetaxel decreased the clonogenic potential (interaction test for SUM149 and SUM159, p

Cyclin-dependent kinases 4 and 6 (CDK4/6) play critical roles in the G1 to S checkpoint of the cell cycle and have been shown to be overactive in several human cancers. Small-molecule inhibitors of CDK4/6 have demonstrated significant efficacy against many solid tumors. Since CDK4/6 inhibition is thought to induce cell cycle arrest at the G1/S checkpoint, much interest has been focused on combining CDK4/6 inhibitors with cytotoxic agents active against the S or M phase of the cell cycle to enhance therapeutic efficacy. However, it remains unclear how best to combine these two classes of drugs to avoid their potentially antagonistic effects. Here, we test various combinations of highly selective and potent CDK4/6 inhibitors with commonly used cytotoxic drugs in several cancer cell lines derived from lung, breast and brain cancers, for their cell-killing effects as compared to monotherapy. All combinations, either concurrent or sequential, failed to enhance cell-killing effects. Importantly, in certain schedules, especially pre-treatment with a CDK4/6 inhibitor, combining these drugs resulted in reduced cytotoxicity of cytotoxic agents. These findings urge cautions when combining these two classes of agents in clinical settings.

A novel LC-MS/MS method was developed for the quantification of the new cyclin dependent kinase inhibitors (CDKIs) palbociclib and ribociclib and the aromatase inhibitor letrozole used in combinatory regimen. The proposed method is appropriate to be applied in clinical practice due to the simple and fast sample preparation based on protein precipitation, the low amount of patient sample necessary for the analysis (10 μL) and the total run time of 6.5 min. It was fully validated according to FDA and EMA guidelines on bioanalytical method validation. The linearity was assessed (R2 within 0.9992-0.9983) over the concentration ranges of 0.3-250 ng/mL for palbociclib, 10-10000 ng/mL for ribociclib and 0.5-500 ng/mL for letrozole that properly cover the therapeutic plasma concentrations. A specific strategy was implemented to reduce the carryover phenomenon, formerly known for these CDKIs. This method was applied to quantify the Cmin of palbociclib, ribociclib and letrozole in plasma samples from patients enrolled in a clinical study. The same set of study samples was analysed twice in separate runs to assess the reproducibility of the method by means of the incurred samples reanalysis. The results corroborated the reliability of the analyte concentrations obtained with the bioanalytical method, already proved by the validation process. The percentage differences were always within ±10% for all the analytes and the R2 of the correlation graph between the two quantifications was equal to 0.9994.

ObjectiveTo determine the transplacental pharmacokinetics of the HIV integrase inhibitor dolutegravir.Study designMaternal-to-fetal transfer across the term human placenta was investigated with the ex-vivo dually perfused cotyledon model, in 5 closed-circuit, recirculating experiments. Dolutegravir was added to a maternal perfusate containing antipyrine, a marker to validate the cotyledon's viability, and 2 g/liter of human albumin.ResultsAfter 3h of recirculating perfusion, the mean (± SD) DTG concentrations in the maternal and in the fetal compartments were respectively 2450 ± 286 ng/mL and 715 ± 369 ng/mL, with a fetal-to-maternal ratio of 34% ± 18% and a clearance index (in comparison with antipyrine transfer) of 79% ± 23%. The mean cotyledon accumulation index was 153% ± 25%.ConclusionFetal transplacental exposure to dolutegravir was considerable as well as accumulation in placental tissue. Whether this may lead to risks for the exposed fetus requires more investigation.

Trazodone is an antidepressant drug with considerable affinity for 5-HT1A receptors and α1-adrenoceptors for which the drug is competitive agonist and antagonist, respectively. In this study, we used cell-attached or whole-cell patch-clamp recordings to characterize the effects of trazodone at somatodendritic 5-HT1A receptors (5-HT1AARs) and α1-adrenoceptors of serotonergic neurons in rodent dorsal raphe slices. To reveal the effects of trazodone at α1-adrenoceptors, the baseline firing of 5-HT neurons was facilitated by applying the selective α1-adrenoceptor agonist phenylephrine at various concentrations. In the absence of phenylephrine, trazodone (1-10 μM) concentration-dependently silenced neurons through activation of 5-HT1AARs. The effect was fully antagonized by the selective 5-HT1A receptor antagonist Way-100635. With 5-HT1A receptors blocked by Way-100635, trazodone (1-10 μM) concentration-dependently inhibited neuron firing facilitated by 1 μM phenylephrine. Parallel rightward shift of dose-response curves for trazodone recorded in higher phenylephrine concentrations (10-100 μM) indicated competitive antagonism at α1-adrenoceptors. Both effects of trazodone were also observed in slices from Tph2-/- mice that lack synthesis of brain serotonin, showing that the activation of 5-HT1AARs was not mediated by endogenous serotonin. In whole-cell recordings, trazodone activated 5-HT1AAR-coupled G protein-activated inwardly-rectifying (GIRK) channel conductance with weak partial agonist efficacy (~35%) compared to that of the full agonist 5-CT. Collectively our data show that trazodone, at concentrations relevant to its clinical effects, exerts weak partial agonism at 5-HT1AARs and disfacilitation of firing through α1-adrenoceptor antagonism. These two actions converge in inhibiting dorsal raphe serotonergic neuron activity, albeit with varying contribution depending on the intensity of α1-adrenoceptor stimulation.

Standard treatment, unfortunately, yields a poor prognosis for patients with primary or metastatic cancers in the central nervous system, indicating a necessity for novel therapeutic agents. Immunotoxins (ITs) are a class of promising therapeutic candidates produced by fusing antibody fragments with toxin moieties. In this study, we investigated if inherent resistance to IT cytotoxicity can be overcome by rational combination with pro-apoptotic enhancers. Therefore, we combined ITs (9.2.27-PE38KDEL or Mel-14-PE38KDEL) targeting chondroitin sulfate proteoglycan 4 (CSPG4) with a panel of Bcl-2 family inhibitors (ABT-737, ABT-263, ABT-199 [Venetoclax], A-1155463, and S63845) against patient-derived glioblastoma, melanoma, and breast cancer cells/cell lines. In vitro cytotoxicity assays demonstrated that the addition of the ABT compounds, specifically ABT-737, sensitized the different tumors to IT treatment, and improved the IC50 values of 9.2.27-PE38KDEL up to >1,000-fold. Mechanistic studies using 9.2.27-PE38KDEL and ABT-737 revealed that increased levels of intracellular IT, processed (active) exotoxin, and PARP cleavage correlated with the enhanced sensitivity to the combination treatment. Furthermore, we confirmed the synergistic effect of 9.2.27-PE38KDEL and ABT-737 combination therapy in orthotopic GBM xenograft and cerebral melanoma metastasis models in nude mice. Our study defines strategies for overcoming IT resistance and enhancing specific antitumor cytotoxicity in primary and metastatic brain tumors.

Purpose A patient-derived xenograft (PDX) model is an in vivo animal model which provides biological and genomic profiles similar to a primary tumor. The characterization of factors that influence the establishment of PDX is crucial. Furthermore, PDX models can provide a platform for chemosensitivity tests to evaluate the effectiveness of a target agent before applying it in clinical trials. Methods We implanted 83 cases of breast cancer into NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic mice, to develop PDX models. Clinicopathological factors of primary tumors were reviewed to identify the factors affecting engraftment success rates. After the establishment of PDX models, we performed olaparib and carboplatin chemosensitivity tests. We used PDX models from triple-negative breast cancer (TNBC) with neoadjuvant chemotherapy and/or germline BRCA1 mutations in chemosensitivity tests. Results The univariate analyses (p

The emergence of Mycobacterium tuberculosis strains resistant to current first-line antibiotic regimens constitutes a major global health threat. New treatments against multidrug-resistant tuberculosis (MDR-TB) are thus eagerly needed in particular in countries with a high MDR-TB prevalence. In this context, macozinone (PBTZ169), a promising drug candidate with an unique mode of action and highly potent in vitro tuberculocidal properties against MDR Mycobacterium strains, has now reached the clinical phase and has been notably tested in healthy male volunteers in Switzerland. To that endeavor, a multiplex UHPLC-MS/MS method has been developed for the sensitive and accurate human plasma levels determination of PBTZ169 along with five metabolites retaining in vitro anti-TB activity. Plasma protein precipitation with methanol was carried out as a simplified sample clean-up procedure followed by direct injection of the undiluted supernatant for the bioanalysis of the six analytes within 5 min, using 1.8 mu m reversed-phase chromatography coupled to triple quadrupole mass spectrometry employing electrospray ionization in the positive mode. Stable isotopically-labelled PBTZ169 was used as internal standard (ISTD), while metabolites could be reliably quantified using two unlabeled chemical analogues selected as ISTD from a large in-house analogous compounds library. The overall methodology was fully validated according to current recommendations (FDA, EMEA) for bioanalytical methods, which include selectivity, carryover, qualitative and quantitative matrix effect, extraction recovery, process efficiency, trueness, precision, accuracy profiles, method and instrument detection limits, integrity to dilution, anticoagulant comparison and short-and long-term stabilities. Stability studies on the reduced metabolite H-2-PBTZ169 have shown no significant impact on the actual PBTZ169 concentrations determined with the proposed assay. This simplified, rapid, sensitive and robust methodology has been applied to the bioanalysis of human plasma samples collected within the frame of a phase I clinical study in healthy volunteers receiving PBTZ169.

BackgroundRoll-out of Integrase Strand Transfer Inhibitors (INSTIs) such as dolutegravir for HIV combination antiretroviral therapy (cART) in sub-Saharan Africa necessitates the development of affordable HIV drug resistance (HIVDR) assays targeting the Integrase gene. We optimised and evaluated an in-house integrase HIV-1 drug resistance assay (IH-Int) and compared it to a commercially available assay, ViroSeq™ Integrase Genotyping kit (VS-Int) amongst HIV-1 clade C infected individuals.MethodsWe used 54 plasma samples from treatment naïve participants and one plasma sample from a patient failing INSTI based cART. Specimens were genotyped using both the VS-Int and IH-Int assays. Stanford HIV drug resistance database were used for integrase resistance interpretation. We compared the major and minor resistance mutations, pairwise nucleotide and amino-acid identity, costs and assay time.ResultsAmong 55 specimens tested with IH-Int, 53 (96.4%) successfully amplified compared to 45/55 (81.8%) for the VS-Int assay. The mean nucleotide and amino acid similarity from 33 paired sequences was 99.8% (SD ± 0.30) and 99.8% (SD ± 0.39) for the IH-Int and VS-Int assay respectively. The reagent cost/sample were 32 USD and 147 USD for IH-Int and VS-Int assay, respectively. All sequenced samples were confirmed as HIV-1 subtype C.ConclusionsThe IH-Int assay had a high amplification success rate and high concordance with the commercial assay. It is significantly cheaper compared to the commercial assay. Our assay has the needed specifications for routine monitoring of participants on Dolutegravir based regimens in Botswana.

Numerous studies have shown that the serotonin(1A) (5-HT1A) receptor is implicated in the pathophysiology and treatment of several psychiatric and neurological disorders. Furthermore, functional imaging studies in a variety of species have demonstrated that 4-(2'-Methoxyphenyl)-1-[2'-(N-2 ''-pyridinyl)-p-[F-18]fluorobenzamidoethylpiperazine ([F-18]MPPF) is a valid and useful PET tracer to visualize the 5HT(1A) receptor. However, to our knowledge, [F-18] MPPF has never been demonstrated in the canine brain. The ability to image the 5HT(1A) receptor with PET in dogs could improve diagnosis and therapy in both canine and human behavioural and neuropsychiatric disorders. To examine the potential use of [F-18]MPPF in dogs, five healthy adult laboratory beagles underwent a 60-minutes dynamic PET scan with [F-18]MPPF while arterial blood samples were taken. For each region of interest, total distribution volume (V-T) and corresponding binding potential (BPND) were calculated using the 1-tissue compartment model (1-TC), 2-Tissue compartment model (2-TC) and Logan plot. The preferred model was chosen based on the goodness-of-fit, calculated with the Akaike information criterium (AIC). Subsequently, the BPND values of the preferred compartment model were compared with the estimated BPND values using three reference tissue models (RTMs): the 2-step simplified reference tissue model (SRTM2), the 2-parameter multilinear reference tissue model (MRTM2) and the Logan reference tissue model. According to the lower AIC values of the 2-TC model compared to the 1-TC in all ROIs, the 2-TC model showed a better fit. Calculating BPND using reference tissue modelling demonstrated high correlation with the BPND obtained by metabolite corrected plasma input 2-TC. This first-indog study indicates the results of a bolus injection with [F-18] MPPF in dogs are consistent with the observations presented in the literature for other animal species and humans. Furthermore, for future experiments, compartmental modelling using invasive blood sampling could be replaced by RTMs, using the cerebellum as reference region.

Although mechanisms of acquired resistance to 1st and 3rd generation EGFR-TKI continue to be elucidated, there have been few clinical investigations into the mechanisms of acquired resistance to the 2nd generation EGFR-TKI afatinib. We analyzed data from 20 patients with advanced lung adenocarcinoma who acquired resistance to afatinib, including resistance during EGFR-TKI re-challenge. We examined EGFR T790M and C797S mutations, BRAF V600E mutation, and MET amplification with the MBP-QP method and with droplet digital PCR using ctDNA and re-biopsy samples obtained before and after afatinib treatment. Just before afatinib treatment, 15 of the 20 patients were T790M negative and five were positive. Among the T790M negative patients, 40.0% (6/15) became positive at the time of PD under afatinib. In patients positive for T790M, changes in T790M allele frequency were correlated with afatinib treatment efficacy. C797S was not detected in any patients just before afatinib treatment, but it appeared after treatment in three patients, although with very low allele frequency. Two of these three patients, although positive for both C797S and T790M, achieved PR to osimertinib. However, PFS of these patients was somewhat shorter than that of patients positive for T790M only. BRAF V600E was detected in one patient at PD under afatinib. MET amplification was not detected in this study. T790M is associated with acquired resistance to afatinib, as with 1st generation EGFR-TKI, but with somewhat lower frequency. The influence of C797S on resistance to afatinib is less than that of T790M, but C797S might cause shorter PFS under osimertinib.

DNA topoisomerase II (topo II) is an essential enzyme that regulates DNA topology by DNA cleavage and re-ligation. In vertebrates, there are two isozymes, α and β. The C-terminal domain (CTD) of the isozymes, which shows a low degree of sequence homology between α and β, is involved in each isozyme-specific intracellular behavior. The CTD of topo IIβ is supposedly involved in topo II regulation. Topo IIβ is maintained in an inactive state in the nucleoli by the binding of RNA to the 50-residue region termed C-terminal regulatory domain (CRD) present in the CTD. Although in vitro biochemical analysis indicates that the CTD of topo IIβ has DNA binding activity, it is unclear whether CTD influences catalytic reaction in the nucleoplasm. Here, we show that the proximal CTD (hereafter referred to as pCTD) of rat topo IIβ, including the CRD, is involved in the catalytic reaction in the nucleoplasm. We identified the pCTD as a domain with DNA binding activity by in vitro catenation assay and electrophoretic mobility shift assay. Fluorescence recovery after photo-bleaching (FRAP) analysis of pCTD-lacking mutant (ΔpCTD) showed higher mobility in nucleoplasm than that of the wild-type enzyme, indicating that the pCTD also affected the nuclear dynamics of topo IIβ. ICRF-193, one of the topo II catalytic inhibitors, induces the formation of closed-clamp intermediates of topo II. Treatment of ΔpCTD with ICRF-193 significantly decreased the efficiency of closed-clamp formation. Altogether, our data indicate that the binding of topo IIβ to DNA through the pCTD is required for the catalytic reaction in the nucleoplasm.

Uganda adopted the integrase inhibitor dolutegravir (DTG) as part its preferred first-line HIV treatment regimen in 2018. Prior to the national rollout, the Uganda Ministry of Health and Clinton Health Access Initiative (CHAI) launched a pilot study in July 2017 aimed at better understanding patients’ and prescribers’ experience and acceptability of DTG. Patients were enrolled in the study if they were newly initiating treatment or switched from an NNRTI regimen due to intolerance. Patients were followed up for 6 months after initiation onto DTG and acceptability and experiences were assessed through questionnaires at one-month and six-month follow-up visits. In addition to acceptability side effects of patients on DTG regimens were assessed. Analysis was conducted using MS Excel and SAS 9.4 and confidence intervals were adjusted for facility level clustering. A total of 365 patients from 6 study sites were enrolled in the study, of whom 50% were treatment-experienced and 50% treatment naïve. 325 patients completed the 6 months of follow-up. Survey results showed a high level of acceptability (more than 90%) of DTG-containing regimens for both categories of patients during the from one-month and six-months interviews. The rate of self-reported side effects amongst patients was 33% overall and higher for experienced (37%) than naïve (29%) patients at 6 months. Although frequencies declined between month-1 and month-6, the changes were not statistically significant. Almost all patients (94%) were virally suppressed at 6 months. Overall, the study findings showed a very high level of acceptability of Dolutegravir-based regimens across both experienced and naïve patients. The overall viral suppression rate in this cohort was 94% at six months of taking DTG-based regimen.

BackgroundWhile the scale-up of HIV services has improved national health management information systems (HMIS), there remain challenges in using routine data to guide the introduction of optimized antiretroviral (ARV) drugs.MethodsBuilding on the recent enhancements to the HMIS in Kenya and coinciding with the introduction of a new ARV regimen, tenofovir+lamivudine+dolutegravir (TLD), we developed and implemented an enhanced data system (EDS) to improve availability of safety and efficacy data among people living with HIV (PLHIV) in Kenya. Using data from one health facility, we showcase how the EDS can be used to monitor ARV transition and identify missed opportunities to transition eligible patients to optimized regimes.ResultsThe EDS was designed to create a comprehensive PLHIV database by triangulating patient-level data from the EMR, the pharmacy ARV dispensing tool (ADT) and HIV viral load (VL) databases. On a monthly basis, the database is de-identified and uploaded into a national data warehouse, with interactive dashboards. Using the EDS, we determined that of the 5,500 PLHIV ≥15 years on first-line ART at one facility, 4,233 (77%) had transitioned to optimized ARVs. Of the 1,267 still on legacy regimens, 459 (36%) were determined to be eligible and prioritized to switch.ConclusionsThis project illustrates how enhancements to the national HMIS can facilitate the use of routine patient-level data to monitor the transition to new ARVs and inform the national HIV response.

Several ultra-sensitive methods for T790M in plasma cell-free DNA (cfDNA) have been developed for lung cancer. The correlation between mutation-allele frequency (MAF) cut-off, drug responsiveness, and outcome prediction is an unmet needs and not fully addressed. An innovative combination of peptide nucleic acid (PNA) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was used to proof of concept for monitoring cfDNA T790M in EGFR-mutant patients. Mutant enrichment by PNA was optimized and the detection limit was evaluated through serial dilutions. The cut-off value was identified by receiver-operating-characteristic (ROC) curve analysis utilizing serial sampled plasmas of patients from EGFR-tyrosine kinase inhibitor (TKI) pretreatment to progressive-disease (PD). Results, comparisons, and objective response rate (ORR) were analyzed in 103 patients' tumor and cfDNA T790M, with 20 of them receiving an additional COBAS test. The detection limit was 0.1% MAF. The cut-off for PD and imminent PD was 15% and 5% with an ROC area under the curve (AUC) of 0.96 and 0.82 in 2 ml plasma. Detection sensitivity of cfDNA T790M was 67.4% and overall concordance was 78.6%. ORR was similar in T790M-positive cfDNA (69.6%) and tumor samples (70.6%) treated with osimertinib. Among 65 T790M-positive tumors, 15 were negative in cfDNA (23.1%). Seven of 38 T790M-positive cfDNA samples were negative in the tumors (18.4%). PNA-MALDI-TOF MS had a higher detection rate than COBAS. In conclusion, identification of T790M cut-off value in cfDNA improves cancer managements. We provide a strategy for optimizing testing utility, flexibility, quality, and cost in the clinical practice.

PARP inhibitors target BRCA mutations and defective homologous recombination repair (HRR) for the treatment of epithelial ovarian cancer (EOC). However, the treatment of HRR-proficient EOC with PARP inhibitors remains challenging. The objective of this study was to determine whether the combination of triapine (ribonucleotide reductase inhibitor), cediranib (vascular endothelial growth factor receptor tyrosine kinase inhibitor), and the PARP inhibitor olaparib synergized against BRCA wild-type and HRR-proficient EOC in xenograft mouse models. In addition, the mechanisms by which cediranib augmented the efficacy of triapine and olaparib were investigated. BRCA-wild type and PARP inhibitor-resistant EOC cell lines were implanted subcutaneously (s.c.) into nude mice or injected intraperitoneally (i.p.) into SCID-Beige mice. Mice were then treated i.p. with olaparib, cediranib, triapine, various double and triple combinations. The volume of s.c tumor in nude mice and the abdominal circumference of SCID-Beige mice were measured to evaluate the effectiveness of the treatment to delay tumor growth and prolong the survival time of mice. In both xenograft mouse models, the combination of triapine, olaparib and cediranib resulted in marked suppression of BRCA-wild type EOC growth and significant prolongation of the survival time of mice, with efficacy greater than any double combinations and single drugs. Furthermore, we identified that cediranib abrogated pro-survival and anti-apoptotic AKT signaling, thereby enhancing the efficacy of triapine and olaparib against BRCA-wild type EOC cells. Taken together, our results demonstrate a proof-of-principle approach and the combination regiment holds promise in treating BRCA-wild type and PARP inhibitor-resistant EOC.

High concentrations of antioxidants in cancer cells are huge obstacle in cancer radiotherapy. Erastin was first discovered as an inducer of iron-dependent cell death called ferroptosis accompanied by antioxidant depletion caused by cystine glutamate antiporter inhibition. Therefore, treatment with erastin is expected to potentially enhance cellular radiosensitivity. In this study, we investigated the influence of treatment with erastin on the radiation efficiency against cancers. The clonogenic ability, glutathione peroxidase 4 (GPX4) expression, and glutathione concentration were evaluated using HeLa and NCI-H1975 adenocarcinoma cell lines treated with erastin and/or X-ray irradiation. For in vivo studies, NCI-H1975 cells were transplanted in the left shoulder of nude mice, and then radiosensitizing effect of erastin and glutathione concentration in the cancer were evaluated. Treatment with erastin induced ferroptosis and decreased the concentration of glutathione and GPX4 protein expression levels in the two tumor cell lines. Moreover, erastin enhanced X-ray irradiation-induced cell death in both human tumor cell lines. Furthermore, erastin treatment of a tumor-transplanted mouse model similarly demonstrated the radiosensitizing effect and decrease in intratumoral glutathione concentration in the in vitro study. In conclusion, our study demonstrated the radiosensitizing effect of erastin on two adenocarcinoma cell lines and the tumor xenograft model accompanied by glutathione depletion, indicating that ferroptosis inducers that reduce glutathione concentration could be applied as a novel cancer therapy in combination with radiotherapy.

The incidence of Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing rapidly in the UK. Patients with HPV-positive OPSCC generally show superior clinical responses relative to HPV-negative patients. We hypothesised that these superior responses could be associated with defective repair of DNA double strand breaks (DSB). The study aimed to determine whether defective DNA repair could be associated with sensitivity to inhibition of DNA repair using the PARP inhibitor Olaparib. Sensitivity to Olaparib, and induction and repair of DNA damage, were assessed in a panel of 8 OPSCC cell-lines, including 2 novel HPV-positive lines. Effects on cell cycle distribution and levels of PARP1 and p53 were quantified. RNA-sequencing was used to assess differences in activity of DNA repair pathways. Two HPV-positive OPSCC lines were sensitive to Olaparib at potentially therapeutic doses (0.1-0.5 μM). Two HPV-negative lines were sensitive at an intermediate dose. Four other lines, derived from HPV-positive and HPV-negative tumours, were resistant to PARP inhibition. Only one cell-line, UPCISCC90, showed results consistent with the original hypothesis i.e. that in HPV-positive cells, treatment with Olaparib would cause accumulation of DSB, resulting in cell cycle arrest. There was no evidence that HPV-positive tumours exhibit defective repair of DSB. However, the data suggest that a subset of OPSCC may be susceptible to PARP-inhibitor based therapy.

While the cerebral autoregulation sufficiently protects subcortical brain regions during hypoxia or asphyxia, the cerebral cortex is not as adequately protected, which suggests that regulation of the cerebral blood flow (CBF) is area-specific. Hypoxia was induced by inhalation of 5% oxygen, for reoxygenation 100% oxygen was used. Cortical and subcortical CBF (by laser Doppler flowmetry), blood gases, mean arterial blood pressure (MABP), heart rate and renal blood flow were constantly monitored. Low dosed urapidil was used for α1A-adrenergic receptor blockade. Western blotting was used to determine adrenergic receptor signalling mediators in brain arterioles. During hypoxia cortical CBF decreased to 72 ± 11% (mean reduction 11 ± 3%, p < 0.001) of baseline, whereas subcortical CBF increased to 168±18% (mean increase 43 ± 5%, p < 0.001). Reoxygenation led to peak CBF of 194 ± 27% in the subcortex, and restored cortical CBF. α1A-Adrenergic blockade led to minor changes in cortical CBF, but massively reduced subcortical CBF during hypoxia and reoxygenation-almost aligning CBF in both brain regions. Correlation analyses revealed that α1A-adrenergic blockade renders all CBF-responses pressure-passive during hypoxia and reoxygenation, and confirmed the necessity of α1A-adrenergic signalling for coupling of CBF-responses to oxygen saturation. Expression levels and activation state of key signalling-mediators of α1-receptors (NOSs, CREB, ERK1/2) did not differ between cortex and subcortex. The dichotomy between subcortical and cortical CBF during hypoxia and reoxygenation critically depends on α1A-adrenergic receptors, but not on differential expression of signalling-mediators: signalling through the α1A-subtype is a prerequisite for cortical/subcortical redistribution of CBF.